Breast cancers are heterogeneous diseases with significant mortality associated with failed treatment, and this is primarily due to the non-specifc nature of most therapies. Indeed, with the notable exception of anti-human epidermal growth factor receptor 2 (HER2) agents, most breast cancer therapies act through generalized toxicity. Triple-negative breast cancers, which are associated with high aggressiveness and poor prognosis, fail to express HER2 and have even less treatment options. Thus, the search for new therapeutic intervention points in breast cancers is a crucial unmet medical need. The tyrosine phosphatase SHP2, encoded by the well-known oncogene PTPN11, is a candidate for targeted cancer therapy. A plethora of existing data demonstrates that aberrant catalytic function of SHP2 is responsible for its oncogenic effects. Recent genetic evidence indicates that SHP2 is indeed a novel potential target in HER2-positive and triple negative breast cancers, as its ablation results in decreased tumor burden and reduced metastatic progression. To further investigate these phenomena, such studies require highly potent, specific, and efficacious SHP2 inhibitors with properties that make them suitable for in vivo experiments. However, none of the reported SHP2 inhibitors fully satisfies these requirements. One of the major obstacles in developing such compounds is the lack of compound selectivity for SHP2 over other tyrosine phosphatases. Another considerable hurdle is presented by the fact that existing SHP2 inhibitors carry a charged phosphotyrosine-mimicking group that precludes sufficient compound exposure in vivo. We have generated novel allosteric inhibitors with excellent selectivity for SHP2. Our compounds do not have such a charged moiety as they do not target the phosphotyrosine binding site in SHP2. Here, we propose to define the basis of SHP2 inhibition by these compounds in order to facilitate chemical optimization of existing lead structures, as well as future drug discovery efforts. Optimized leads with further increased potency/ efficacy and suitable drug metabolism and pharmacokinetics (DMPK) properties will be utilized to fully validate SHP2 as drug target for breast cancer therapy. The overarching objectives of this proposal therefore are to demonstrate the efficacy of our SHP2 inhibitors in cellular breast cancer models, to optimize the agents as potent drug like molecules, and finally to demonstrate their potential as lead agents in in vivo models of breast cancers. To achieve these objectives, we will use X-Ray crystallography and NMR spectroscopy to identify the novel allosteric site(s) targeted by our lead compounds (Aim 1). We will chemically optimize the SHP2 inhibitors for improved solubility, efficacy, and DMPK properties to make them suitable for in vivo studies (Aim 2). Finally, we will test optimized SHP2 inhibitors for their potential to inhibit a) SHP2-dependent signaling in breast cancer cells, b) invasion and proliferation of breast cancer cells in three-dimensional culture and in vivo, and c) tumor maintenance, growth, and progression to lung metastases in triple negative breast cancer mouse models (Aim 3).